Our discovery of synthetic small molecule, pluripotin, and its mechanistic characterizations provided a powerful chemical tool for mESC culture and revealed a novel insight into ESC self-renewal: instead of activating dispensable genes/pathways by exogenous growth factors, the key to achieve a basic/fundamental self- renewal state of ESCs is to inhibit various intrinsic differentiation-inducing genes. Because the hESC field is still at its infancy (e.g. a robust self-renewal condition for hESCs would be highly desirable), unbiased and functional discovery approach would be highly suited, e.g. providing tools and laying ground for hypotheses. To address the hypothesis whether this notion is also true for human ESCs, i.e. whether small molecules can be identified that inhibit differentiation of hESCs in the absence of growth factors/cytokines for long-term self- renewal of hESCs (in a way conceptually similar to the function/mechanism of pluripotin in mESCs), we propose to screen 100,000 diverse and discrete compounds to identify small molecules that can maintain self- renewal of hESCs in the chemically defined media in the absence of bFGF and other growth factors. We will further confirm and characterize their effects and activities via various in-depth cellular/biochemical assays, and carry out structure-activity-relationship (SAR) studies of the selected hit compounds to optimize their potency and specificity. Collectively, the studies described in this proposal will speed up understanding of the self-renewal and differentiation mechanisms, allow much more consistency in hESC culture, provide new avenues in practical applications of hESCs in research and in regenerative medicine, and facilitate defining and controlling signaling inputs that direct self-renewal or differentiation of hESCs. PUBLIC HEALTH RELEVANCE: To address whether small molecules can be identified that inhibit differentiation of hESCs in the absence of growth factors/cytokines for long-term self-renewal of hESCs (in a way conceptually similar to the function/mechanism of pluripotin in mESCs), we propose to screen 100,000 diverse and discrete compounds to identify small molecules that can maintain self-renewal of hESCs in the chemically defined media in the absence of bFGF and other growth factors. Collectively, the studies described in this proposal will provide novel chemical tools for better understanding and controlling hESC self-renewal, and may ultimately allow development of therapeutics employing hESCs for treating diseases.